1. Field of the Invention
The present invention relates to a method of discriminating a target base carried by a DNA and an allele specific primer for use in the same.
2. Description of the Related Art
SNP is a polymorphism which occurs most frequently among gene polymorphisms, and is believed to appear in human genomes with an incidence of about 0.1%.
As a matter of fact, presence of SNPs of as many as over three million has been hitherto elucidated, suggesting that SNP may be very useful as a marker for genetic tests.
Investigations on relationship between SNPs and diseases carried out so far have revealed close relationship between diseases such as diabetes and hypertension, and SNP.
Known techniques for carrying out SNP discrimination include techniques in which a primer extension reaction (including PCR reaction) by an allele specific primer is utilized. A primer which yields a marked difference in efficiency of the primer extension reactions depending on the base type at targeted SNP is referred to as the allele specific primer. Therefore, the base type at the target SNP can be readily specified, for example, by carrying out a PCR reaction using an allele specific primer, and analyzing the amount of the PCR product.
In general, allele specific primers are simple origo DNAs which are not anyhow modified especially.
For analysis of the amount of PCR product, so called general electrophoretic method may be used. Thus, SNP discrimination techniques with an allele specific primer may be referred to as being extremely advantageous in terms of cost, reaction time, convenience of operation and the like.
With respect to methods of analysis, in addition to the aforementioned electrophoretic method, detection by QCM or SPR is also enabled when a solid phase reaction is carried out.
More recently, methods of detecting PPi (pyrophosphate) that is a byproduct of a primer extension reaction utilizing a luciferase reaction have been also developed. Accordingly, approaches to simplification and acceleration of SNP discrimination with an allele specific primer have been elaborately attempted all over the world.
Sequence design of an allele specific primer is a very important factor for its ability to discriminate SNP.
In the most classical example, 3′ end base corresponds to the target SNP base (i.e., complementary to any one of predicted base types at the target SNP), and other sequence is completely complementary to the target DNA sequence. However, in this case, problems of the false positive may be raised unless reaction conditions are strictly defined such as the reaction time and temperature, or concentration of dNTPs used in the reaction, as well as cycle number when a PCR method is employed. In other words, fundamentally, although a primer extension reaction must not be caused from a base corresponding to SNP of the primer 3′ end when it is noncomplementary to the target SNP base of a sample, the reaction is caused in many cases.
In order to solve the aforementioned problems in connection with the false positive, several novel allele specific primers have been developed so far.
An allele specific primer proposed in US Patent Publication No. 2003/0049628 is a developed allele specific primer in which the 3′ end base is a base corresponding to SNP, and the third base from the 3′ end was intentionally made noncomplementary to a base adjacent to on the 3′ side of the target SNP base with two bases apart.
US Patent Publication No. 2003/0148301 proposes an allele specific primer in which the second base from the 3′ end is a base corresponding to SNP, and the third base from the 3′ end base is intentionally made noncomplementary to a base adjacent to on the 3′ side of the target SNP base. In this instance, KOD polymerase that is 3′→5′ exo+polymerase is characteristically utilized.
US Patent Publication No. 2004/0197803 proposes an allele specific primer in which 3′ end base is a base corresponding to SNP, and the second and the third bases from the 3′ end are intentionally made noncomplementary to a base adjacent to on the 3′ side of the target SNP base and a base adjacent with one base spaced apart, respectively. Among them, the allele specific primer proposed in US Patent Publication No. 2004/0197803 involves low possibility of the false positive, in particular.
More specifically, the allele specific primer disclosed in US Patent Publication No. 2004/0197803 which was designed as described above may construct a loop structure, as shown in FIGS. 1A and 1B, because only the second and the third bases from the 3′ end are noncomplementary to the target DNA sequence (in the Figure, noncomplementary base pair is denoted by “x”) when the base corresponding to SNP positioned to its 3′ end (in the Figure, denoted by “S′”) is complementary to the target SNP base (in the Figure, denoted by “S”) (FIG. 1A). Consequently, the polymerase is efficiently bound to cause a primer extension reaction.
To the contrary, when the base corresponding to SNP positioned to its 3′ end is noncomplementary to the target SNP base, all three bases at the 3′ end become noncomplementary, and branched structure may be constructed (FIG. 1B). In this case, the primer extension reaction is hardly caused because binding efficiency of polymerase is believed to be extremely low. Therefore, the allele specific primer proposed in US Patent Publication No. 2004/0197803 shall involve extremely low possibility of the false positive as described above.
In addition, the following documents can be referred to as relevant documents to the present invention.
Japanese Patent Provisional Publication No. 2004-248635 discloses a PCR primer for identification of rice variety in which 3′ end corresponds to the SNP site to be discriminated, and the third base from the 3′ end is substituted from a sequence that is complementary to the template sequence to be annealed, with the substitution being G to A, A to C, T to G, or C to A.
Further, paragraph No.0040 of Japanese Patent Provisional Publication No. 2004-248635 discloses that “In one aspect, the present invention provides a primer in which the first, the second, the third or the fourth base from the 3′ end was substituted. This substitution may be just alone, or a combination of two or more. Preferably, only the third base from the 3′ end is substituted. This substitution of the primer may be any arbitrary substitution, but is preferably a substitution of G to T, A to C, T to G, or C to A.”.